The strict regulation of energy metabolism and cell proliferation is critical for the normal function of all eukaryotic life. Improper regulation of thse fundamental cellular processes is associated with multiple disease states, including heart disease and cancer. Examples include shifting the sources of ATP in cells as a result of the Warburg effect or hypoxia, resulting from solid tumor growth exceeding vascularization or cardiac insufficiency. ADP-ribosylation factor like-2 (Arl2), a member of the Arf family of regulatory GTPases, is an ancient and ubiquitous eukaryotic cell regulator that is involved in the maintenance of cellular ATP levels, the progression of cell division and the regulation of transcriptional activators involved in cell proliferation. Maintaining the necessary levels of activated Arl2 within a cell is crucial for the proper regulation of these essential processes. However, the biological mechanisms controlling Arl2 activity are unknown. The majority of cellular Arl2 is in a cytosolic complex with the tubulin-specific co-chaperone, cofactor D (TBCD). We will perform in vitro biochemical analyses to test the hypothesis that dissociation from TBCD is a critical, regulated step in Arl2 function in cells. Additionally, we will test the model that Arl2/TBCD is also an active complex required for proper regulation of centrosomal functions and microtubule dynamics. In specific aim 1, we will utilize recombinant preparations of Arl2 and TBCD to identify the mechanisms regulating assembly/disassembly of the Arl2/TBCD complex. In specific aim 2, we will use cell-based assays to determine the importance of Arl2 and TBCD interaction in regulating centrosomal and microtubule functions. The results of this study will provide fundamental groundwork in our long-term goal of explaining the mechanistic roles of Arl2 as a ubiquitous regulator that is indispensable to the regulation of several essential cellula processes. Further insight into the mechanisms underlying Arl2 regulation will ultimately lead to a better understanding of fundamental eukaryotic functions, such as energy metabolism and cell proliferation.